This proposal is a request for continuation of support of our research on the molecular nature and replication of extrachromosomal elements (plasmids) in bacteria using a number of physical, biochemical, and genetic methods. In previous work we have established a detailed physical map of the transmissible drug resistance (R) plasmid NR1 and localized the genes and functions required for autonomous replication to a 2.2 kilobase segment. For stable inheritance, however, plasmid derivatives require another function (stb) which must be present in cis, suggesting that it plays a structural role in partitioning plasmid molecules at cell division. The specific aims of our research plan concern four aspects of the organization and control of the expression of the replication genes and functions of the R plasmid NR1. (i) The functional role of RNA transcripts from the replication region; (ii) The control of the expression of replication genes and the role of corresponding gene products in plasmid replication; (iii) The mechanism of plasmid copy number control, incompatibility, and the initiation of replication at the origin; (iv) The mechanism by which the stb locus mediates stable plasmid inheritance. This research will contribute in a fundamental way to our understanding of the structure, function, and replication of R plasmids which will be of importance in combatting the spread of multiple drug resistance in bacteria. An understanding of the mechanism of DNA replication and its control will not only be an important advance in its own right, but also will be of fundamental importance in the understanding of cell growth and development. R plasmid replication and drug resistance gene amplification are especially relevant to these phenomena, since the components of R plasmids are controlled by a variety of regulatory mechanisms.